The long-term goal of the proposed research is to understand cellular Zn2+ homeostasis in the brain. Perturbations of Zn2+ homeostasis have been implicated in selective neuronal death following transient brain ischemia and in the pathogenesis of Alzheimer's disease. Thus, a better understanding of Zn2+ homeostasis in the brain will aid in the clarification of the role of Zn2+ in ischemic brain injury and Alzheimer's disease. Yet, homeostasis of Zn2+ in the brain is poorly understood. In previous studies, we have characterized a robust mechanism of plasma membrane Zn2+ transport in cortical neurons that shows a striking dependence on pH. Several lines of experimental evidence suggest that pH plays a very fundamental role in the plasma membrane transport of Zn2+ and other transition elements. Unfortunately, the mechanism(s) underlying pH effects are still unclear. The next logical step to elucidating these mechanisms in neurons now hinges on the identification of the responsible protein(s). The compartmentalization of extrasynaptic Zn2+ in neurons is a newly emerging area of exploration. This laboratory has used zinquin (a Zn2+ specific fluorescent dye) to demonstrate the extrasynaptic compartmentalization of Zn2+ in neurons (see preliminary studies p. 21). The next logical step to understanding the significance of the extrasynaptic compartmentalization of Zn2+ in neurons is to identify the cytoplasmic organelles that sequester Zn2+. Building upon our previous studies and those of others, the research plan described in this grant application outlines the experiments that will take these next logical steps and advance our research effort toward accomplishing our long-term goal. The research plan is organized into two specific aims. Specific Aim 1: Mechanism(s) of plasma membrane Zn2+ transport. Which protein(s) is/are responsible for pH dependent plasma membrane Zn2+ transport in cortical neurons? Specific Aim 2: Extrasynaptic Zn2+ compartmentalization. What is the identity of Zn2+ containing compartments in the cell body and processes of cortical neurons?